Triterpenes are terpenes consisting of six isoprene units having a molecular formula C30H48. Animals, plants and fungi (e.g. yeast), create triterpenes such as squalene and botryococcene. In recent years, studies have been conducted exploring the uses of triterpenes as biofuels and petroleum chemicals.
Triterpenes may be methylated, for example in the form of mono- and di-methylated forms including mono- and di-methylated forms of squalene and botryococcene to name two, as well as other triterpenes. In nature, for example, animal, plant and fungi, triterpenes metabolism occurs in the cytoplasm utilizing the mevalonate (MVA) pathway. FIG. 1 is a schematic showing typical triterpene metabolism occurring in the cytoplasm of animal plant and fungi cells (left portion, FIG. 1). In nature, and in particular in plant cells having chloroplast, monoterpenes and diterpenes are produced in chloroplasts using the methyl erythrithol phosphate (MEP) pathway, for example as shown in the right side of FIG. 1. However, the MEP pathway occurs exclusively in the chloroplast compartment and is responsible for monoterpene, diterpene and polyterpene (carotenoids and phytol) biosynthesis but not triterpene biosynthesis.
Botryococcus braunii accumulates very high levels of methylated triterpenes, compounds that contribute to the buoyancy of the algae and serve as high-valued feedstocks for the petrochemical and chemical industries. Three SAM-dependent methyltransferases catalyzing successive and regio-specific methylations of either squalene or botryococcene are identified. Methylation of the triterpene analogs squalene and botryococcene requires distinct methyltransferases. The observed substrate selectivity and successive cycles of regio-specific catalysis by triterpene methyltransferases from Botryococcus braunii provides evidence that further chemical diversification is achievable.
Botryococcus braunii is a colony-forming, freshwater green algae reported to accumulate 30 to 86% of its dry weight as hydrocarbon oils (1) Three distinct races of B. braunii have been described based on the types of hydrocarbons that each accumulates (2) Race A accumulates fatty acid-derived alkadienes and alkatrienes (3), race L accumulates the tetraterpene lycopadiene (4), and race B accumulates triterpenes, predominately botryococcene, squalene and their methylated derivatives (5) The oils accumulate both in intracellular oil bodies and in association with an extracellular matrix (6), which in race B consists largely of long-chain, cross-linked biopolymers formed in part from acetalization of polymethylsqualene diols (7) Di- and tetra-methylated botryococcenes are generally the most abundant triterpenes accumulating in race B with smaller amounts of tetramethylated-squalene (8) and other structural derivatives of squalene and botryococcene that range from C31 to C37 accumulating to various levels in different strains and in response to variable culture conditions (9) Other polymethylated derivatives such as diepoxy-tetramethylsqualene (10), botryolins (11), and brauixanthins (12) have also been reported.
B. braunii race B has received significant attention because it is considered an ancient algal species dating back at least 500 MYA and is one of the few organisms known to have directly contributed to the existing oil and coal shale deposits found on Earth (13-15), accounting for up to 1.4% of the total hydrocarbon content in oil shales (16) Secondly, because the hydrocarbon oils of B. braunii race B are readily converted to starting materials for industrial chemical manufacturing and high quality fuels under standard hydrocracking/distillation conditions in yields approaching 97% (17), race B has been considered a potential production host for renewable petrochemicals and biofuels. However, the slow growth habit of B. braunii poses serious limitations to its suitability as a robust biofuel production system.
B. braunii race B accumulates triterpene oils in excess of 30% of its dry weight. The composition of the triterpene oils is dominated by di-methylated to tetra-methylated forms of botryococcene and squalene. While unusual mechanisms for the biosynthesis of the botryococcene and squalene were recently described, the enzyme(s) responsible for decorating these triterpene scaffolds with methyl substituents were unknown. A transcriptome of B. braunii was screened computationally assuming that the triterpene methyltransferases (TMTs) might resemble the SAM-dependent enzymes described for methylating the side chain of sterols. Six sterol methyltransferase-like genes were isolated and functionally characterized. Three of these genes when co-expressed in yeast with complementary squalene synthase or botryococcene synthase expression cassettes resulted in the accumulation of mono- and di-methylated forms of both triterpene scaffolds. Surprisingly, TMT-1 and TMT-2 exhibited preference for squalene as the methyl acceptor substrate, while TMT-3 showed a preference for botryococcene as its methyl acceptor substrate. These in vivo preferences were confirmed with in vitro assays utilizing microsomal preparations from yeast over-expressing the respective genes, which encode for membrane associated enzymes. Structural examination of the in vivo yeast generated mono- and di-methylated products by NMR identified terminal carbons, C3 and C22/C20, as the atomic acceptor sites for the methyl additions to squalene and botryococcene, respectively. These sites were identical to those previously reported for the triterpenes extracted from the algae themselves. The availability of closely related triterpene methyltransferases exhibiting distinct substrate specificities and successive catalytic activities provides an important tool for investigating the molecular mechanisms responsible for the specificities exhibited by these unique enzymes.
As previously noted, B. braunii has attracted considerable interest because it reportedly accumulates hydrocarbon oils from 30 to 86% (1) of its dry weight and because these oils are considered progenitors to oil and coal shale deposits (2-4). While all B. braunii are morphologically similar, three distinct chemotypes of B. braunii have been reported depending on the type of hydrocarbons each accumulates (5). Race A accumulates fatty acid-derived alkadienes and alkatrienes (6); race L accumulates the tetraterpene lycopadiene (7); and race B amasses the linear triterpenes, botryococcene, squalene, and their methylated derivatives (8). Di- and tetra-methylated botryococcenes are generally the most abundant oils accumulating in race B (9). However, lower amounts of tetramethylated-squalene (10) and variable amounts of other structural derivatives of botryococcene ranging from C31 to C37 accumulate to various levels in different race B strains and in response to variable culture conditions (9,11). The oils accumulate both in intracellular oil bodies and in association with an extracellular matrix (12), which in race B consists mainly of long-chain, cross-linked polyacetals formed in large part from acetalization of polymethylsqualene diols that account for approximately 10% of the dry weight (13). Other polymethylsqualene derivatives have been described in race B, such as diepoxy-tetramethylsqualene (14), botryolins (15), and braunixanthins (16). The linear triterpenes, botryococcene, squalene, and their methylated derivatives, are hence common components of B. braunii race B and make up a large proportion of its total biomass.
A unique mechanism for botryococcene biosynthesis was recently described by Niehaus et al. (17), in which two squalene synthase-like (SSL) enzymes perform the successive half-reactions that are normally catalyzed by a single enzyme in the case of squalene synthase. SSL-1 uses farnesyl diphosphate (FPP) as a substrate to catalyze the production of pre-squalene diphosphate (PSPP), which a second enzyme, SSL-3, converts to botryococcene in an NADPH-dependent manner. A third enzyme, SSL-2, catalyzes the biosynthesis of squalene from PSPP produced by SSL-1 but cannot efficiently use FPP as a substrate. Overall, it was suggested that the squalene and botryococcene produced by the SSL enzymes were channeled into the production of the liquid oils and the biosynthesis of squalene derivatives, such as the extracellular matrix (17), while the conventional B. braunii squalene synthase (18) appears to synthesize squalene destined for sterol biosynthesis.
It is not botryococcene and squalene, however, that accumulate to substantial levels in this algae, but the methylated forms of these triterpenes. For instance, while the liquid oil content of B. braunii race B is composed primarily of botryococcenes, generally less than 1% is in the non-methylated C30 form and the majority is dominated by di-methylated and tetramethylated forms, depending on the strain and culture conditions (9, Metzger, 1983 #102,11). Essentially all the squalene that accumulates is in methylated forms, accumulating in the oil fraction (less than 5% of the total oil (19) or incorporated into a variety of other B. braunii natural products (13-16). Because B. braunii race B accumulates 30% or more of its dry weight as these triterpene components, one can estimate that the methylated triterpenes can account for up to 25% of the total algal biomass dry weight and contribute directly to the buoyancy that distinguishes these algal colonies. Unlike many green algae that are flagellated and phototaxic (20), the buoyance characteristic of Botryococcus provides a means for it to float in its normal aqueous habitats and to intercept a greater amount of photosynthetic light. In addition to these purported physiological roles, the methylated forms of botryococcene and squalene enhance their utility as feedstocks for petrochemical processing and chemical manufacturing. The increased branching evident in the methylated triterpenes improves their hydrocracking to chemical species of value for the synthesis of industrial polymers and other commodity based chemicals (21) and yields high quality gasoline, kerosene and diesel fuels upon distillation (22).
While the unique mechanisms for C30 botryococcene and squalene biosynthesis in Botryococcus braunii have been elucidated (17), the specific mechanism(s) by which these triterpenes are methylated was unclear at the start of this work. Small molecule methylation has been extensively characterized for many diverse compounds and typically consists of a methyltransferase (MT) that utilizes the universal methyl-donor S-adenosyl methionine (SAM), and exhibits variable degrees of selectivity for a wide range of methyl acceptor molecules (24). MTs are also distinguished as C-, O-, N-, S- or halide methyltransferases, an indication of the methylation target within the acceptor substrate. While MTs may only share limited overall amino acid sequence similarities, domains responsible for SAM binding appear to be broadly conserved and highly conserved structural folds have served to associate MTs into five distinct Classes. Most of the small molecule MTs fall into Class 1, but do not appear to cluster phylogenetically based on their target site (i.e. methylation of carbon versus nitrogen) or the particular chemical class of the methyl acceptor substrate. An indole alkaloid MT, for instance, shows closer sequence similarity to a tocopherol MT rather than any other alkaloid specific MTs. Clustering in this instance appears more related to the evolutionary origins of the MTs and the propensity of MTs to undergo neofunctionalization. There remains a need in the art to harness the unique oil biosynthesis capacity for use in a system that allows for rapid and higher yield production.
There remains a need in the art to harness this unique oil biosynthetic capacity for use in a system that allows for more rapid and higher yield production.